Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and pseudomonas toxin have been coupled to antibodies or receptor binding ligands to generate cell-type-specific-killing reagents (Youle et al., 1980, Proc Natl Acad Sci USA 77:5483-5486; Gilliland et al., 1980, Proc Natl Acad Sci USA 77:4539-4543; Krolick et al., 1980, Proc Natl Acad Sci USA 77:5419-5423). Notwithstanding the fact that the cell-recognition moiety is not always an antibody, these directed toxins are generally known as immunotoxins (ITs). These hybrid proteins kill tumor cells, for example, which express the receptor that the antibody or ligand portion of the molecule recognizes.
Under appropriate conditions, conferred by the particular receptor system, the toxin enters the cytosol, inactivates the protein synthesis machinery and causes death of the target cell. Immunotoxins are highly cytotoxic to cancer cells growing in cell culture and animal models demonstrate the potential of these reagents to treat blood borne malignancies as well as solid tumors in restricted compartments such as the intraperitoneal cavity (reviewed in Griffin et al., 1988, Immunotoxins. Boston/Dordrecht/Lancaster, Kluwer Academic Publishers, p 433; Vitetta et al., 1987, Science 238:1098; Fitzgerald et al., 1989, J. Natl. Cancer Inst. 81:1455).
The injection of ITs containing plant or bacterial proteins into patients was anticipated to elicit an antibody response that would present a major obstacle to the successful application of this technology. Indeed, immune responses against murine monoclonal antibodies (Sawler et al., 1985, J. Immunol. 135:1530-1535; Schroffet et al., 1985, Cancer Res. 45:879-885) and anti-toxin antibodies have been detected in both animals and humans treated with ITs (Rybak et al., 1991, Immunol. and Allergy Clinics of North America 11:2, 359-380; Harkonen et al., 1987, Cancer Res. 47:1377-1385; Hertler, A. (1988) in Immunotoxins (Kluwer Academic Publishers, Boston/Dordrecht/Lancaster,), 475). Although advances in protein design techniques promise to alleviate some of the immunogenicity associated with the antibody portion of ITs (Bird et al., 1988, Science 242:423; Huston et al., 1988, Proc Natl Acad Sci USA 85:5879; Ward et al., 1989, Nature 341:544), no solution has been forthcoming for the immunogenicity of the toxin other than immunosuppression of the patients (Khazaeli et al., 1988, Proceedings of AACR 29:418). Thus, there has been a continuing need for methods and compositions that would reduce the immunogenicity of the toxic moiety of cytotoxic reagents that selectively kill cells having a given surface marker.
In that regard a great deal of effort is being directed toward obtaining chimeric (Boulianne et al., 1985, Nature 643-646; Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855) or humanized antibodies (Jones et al., 1986, Nature (London) 314:522-525) for human therapeutics. Chimeric antibodies combine the variable region binding domain of a murine antibody with human antibody constant regions. "Humanized" antibodies contain only murine complementarity-determining regions combined with human variable region frameworks and human constant regions.
Human transferrin (Tfn) is a serum glycoprotein that binds and delivers iron to cells by receptor mediated endocytosis reviewed in Youle et al., 1987, Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer. Oxford, Oxford University Press. After relinquishing its iron, apo-Tfn-receptor recycles to the cell surface where apo-Tfn is released to continue the cycle. Monoclonal antibodies originally isolated based upon selectivity for tumor cells have been found to react with the human transferrin receptor. Transferrin (Raso et al., 1984, J Biol Chem. 259:1143-1149; O'Keefe et al., 1985, J Biol Chem. 260:932-937.) Or antibodies to the Tfn-receptor (Pirker et al., 1985, Cancer Res. 45:751-757; FitzGerald et al., 1983, Proc Natl Acad Sci USA 80:4134; Scott et al., 1987, J Natl Cancer Inst. 79(5):1163-1172; Trowbridge et al., 1981, Nature 294:171-173) linked to toxic proteins have resulted in highly cytotoxic conjugates specifically toxic to cancer cells in vitro and in vitro (Marks et al., 1990, Cancer Res. 50:288-292.; Scott et al., 1987, J Natl Cancer Inst. 79(5):1163-1172; FitzGerald et al., 1987, Cancer Res. 47:111407-1410).
Toxic ribosome inactivating proteins from plants inactivate protein synthesis by enzymatically cleaving a single N-glycosidic bond of the 28S ribosomal RNA (Endo et al., 1987, J Biol Chem. 262:8128-8130). Other cytotoxic proteins that inactivate ribosomes include .alpha.-sarcin, which is produced by a fungus (Endo et al., 1982, J Biol Chem. 257:9054-9060; Endo et al., 1983, J Biol Chem. 258:2662-2667.) and cloacin DF13, a plasmid encoded bacteriocin (DeGraaf et al., 1977, Eur J. Biochem. 73:107-114) both of which have ribonuclease (RNase) activity.
Analogous toxic mammalian proteins have not been described, but some members of the ribonuclease superfamily in mammals have interesting, possibly related, biological properties. Studies have demonstrated the cytotoxic properties of the ribonuclease superfamily. The bacterial cytotoxins colicin E3 and colicin Df13 target ribosomal RNA (Konishi, J., 1982, Rev Microbiol 36:125-144) and recently a bacterial RNase has been fused to the gene for pseudomonas exotoxin A to create a new chimeric toxin (Prior et al., 1991, Cell 64:1017-1023). The fungal toxin (.alpha.-sarcin expresses RNase activity (Endo, T. & Wool, I., 1982, J. Biol. Chem. 257:9054-9060; Endo et al., 1983, J. Biol. Chem. 258:2662-2667) and in plants normal pollen function in Nicotiana alata is aborted by RNase activity on pollen tube ribosomal RNA (McClure et al., 1990, Nature 347:757-760). The cytotoxicity of RNase A towards tumor cells is well documented from studies performed in the 1960s and 1970s and reviewed in (Roth, J., 1963, Cancer Res. 23:657-666). The relevance of these early studies is underscored by a recent discovery that the anti-tumor protein from oocytes of Rana pipiens has homology to RNase A (Ardelt et al., 1991, J. Biol. Chem. 256:245-251). Furthermore, human serum contains several RNases (Reddi, E., 1975, Biochem. Biophys. Res. Commun 67:110-118, Blank et al., Human body fluid ribonucleases: detection, interrelationships and significance 1-203-209 (IRL Press, London, 1981)) that are expressed in a tissue specific manner. The function of these extracellular RNases are not known but the discovery that proteins involved in the host defense activity of the eosinophil are homologous to RNases and express RNase activity (Gleich et al., 1986, Proc. Natl. Acad. Sci., USA 83:3146-3150; Slifman et al., 1986, J. Immunol, 137:2913-2917) suggests the intriguing possibility that human serum RNases also may have host defense activities.
The human serum ribonuclease angiogenin (Ang) is a human protein with homology to pancreatic RNase (Fett et al., 1985, Biochemistry 24:5480-5485) and RNase activity albeit different than that of the pancreatic enzyme (Rybak et al., 1988, Biochemistry 27:2288-2294; Shapiro et al., 1986, Biochemistry 25:3527-3531). While the active site residues are conserved between Ang and pancreatic RNase, Ang has very little activity toward standard substrates for the pancreatic enzyme (Shapiro et al., 1986, Biochemistry 25:3527-3531).
Studies on in vitro protein synthesis demonstrated that angiogenin inhibited the translational capacity of the rabbit reticulocyte lysate (St. Clair et al., 1987, Proc., Natl. Acad. Sci. USA 84, 8330-8334). Although it was shown that a ribonucleolytic activity of Ang was responsible for this inhibition, no cleavage of ribosomal RNAs could be demonstrated at concentrations of the enzyme that completely inhibited protein synthesis. This was markedly different from pancreatic RNase that inhibited protein synthesis by degrading the major ribosomal and lysate RNAS. These results coupled with the observation that the base cleavage specificity of Ang and pancreatic RNase were the same toward 5S ribosomal RNAs (Rybak et al., 1988, Biochemistry 25:3527-3531) suggested that the ill vivo substrate for Ang was a unique RNA molecule.
Ang is also a potent inhibitor of protein synthesis in cell free extracts (St. Clair et al., 1987, Proc. Natl. Acad. Sci. USA 84,8330-8334) and when directly injected into Xenopus oocytes. Bacterially derived recombinant Ang was injected into Xenopus oocytes and also shown to inhibit protein synthesis without degradation of oocyte ribosomal RNA. Indeed further studies identified tRNA as the intracellular substrate degraded by Ang to inhibit protein synthesis. In a living cell, Ang was as toxic as a fungal toxin (.alpha.-sarcin) currently being used as the toxic moiety in an immunotoxin construct (Wawrzynczak et al., Cytotoxic and Pharmacokinetic Properties of an Immunotoxin made with the ribosome-inactivating Protein Alpha Sarcin from Aspergillus giganteus (Lake Buena Vista, Fla., 1990)). Ang is not cytotoxic toward a wide variety of cultured cells and is normally present in human plasma (Shapiro et al., 1987, Biochemistry 26:5141-5146).
Cytotoxic eosinophil granule proteins also have been reported to have RNase activity (Slifman et al., 1986, J. Immunol. 137(9):2913-2917; Gullberg et al., 1986, Biophys Biochem Res Comm. 139(3):1239-1242), and the sequence of human eosinophil-derived neurotoxin (EDN) is identical to that of the nonsecretory ribonuclease from human urine (Beintema et al., 1988, Biochemistry 27:4530-38). The present inventors have found that eosinophil proteins are also potent inhibitors of cell-free protein synthesis. In addition, antitumor (Vescia et al., 1980, Cancer Res. 40:3740-3744; Matousek, J., 1973, Experientia 29:858-859) and antispermatogenic action (Dostal et al., 1973, J. Reprod., Fert. 34:197-200) have been reported for bovine seminal ribonuclease.